The invention relates to haematological analyses.
More particularly, it concerns a reagent for determination of leucocytes and measurement of haemoglobin in a sample of blood.
The invention also aims to provide such a reagent which permits a leucocytic subpopulation to be identified, in particular the basophil polymorphonuclear leucocytes.
The determination of leucocytes, in particular of certain leucocytic subpopulations, as well as the measurement of the haemoglobin concentration of the erythrocytes or red corpuscles are very important for diagnosis in human pathology.
It will be recalled here that the leucocytes, or human white corpuscles, are divided into five subpopulations, namely three main subpopulations: the lymphocytes, monocytes and polymorphonuclear leucocytes or granulocytes, with the latter being themselves subdivided into neutrophils, eosinophils and basophils, according to the characteristics of their cytoplasmic granules.
Determination of the total leucocytes as well as identification of their various subpopulations are achieved by means of traditional techniques of microscopic observation or by more modern techniques, mainly based on the measurement of variations in resistivity (WO 84/03771) or on optical diffraction (U.S. Pat. No. 3,740,143), which have been developed for specific automatic appliances.
For the identification of the leucocytic subpopulations the numeration of the basophil granulocytes is particularly tricky, considering that this population represents, in a healthy individual, only 0.5% to 1% of the total leucocytic population.
An increase in this basophil population is observed, attaining a population of 2 to 3% by weight, in the course of allergic reactions. Amongst the infections, tuberculosis and varicella can bring about basophilia, as can myxoedema and hyperlipidaemias amongst the metabolic diseases. Consequently, numeration of the basophil granulocytes assumes particular importance.
Patent FR 90 01660 and its equivalent U.S. Pat. No. 5,196,346 describe a reagent that preserves the basophil granulocytes in such a way so as to permit determination thereof by measurement of resistivity. However, this reagent does not permit the measurement of haemoglobin.
It will be recalled that haemoglobin is a chromoprotein contained in the red corpuscles of the blood or erythrocytes.
Measurement of the haemoglobin concentration therefore necessitates the use of a reagent for cellular lysis that is capable of causing lysis of the erythrocytes in order to liberate the haemoglobin for measurement thereof.
For this purpose it is known to use reagents containing at least one detergent and cyanide ions which are capable of carrying out conversion of the haemoglobin into a chromogenic compound in order to permit determination thereof by colorimetry measurement.
A cyanic reagent of this type is described in patents U.S. Pat. No. 3,874,852 and U.S. Pat. No. 3,854,914.
However, these reagents have the principal drawback of using cyanide. Moreover, they do not permit identification and quantification of the leucocytic subpopulations contained in the sample of blood to be analysed.
It should be noted that reagents that do not contain cyanide and that permit determination of leucocytes in addition to measurement of haemoglobin have already been proposed in the prior art.
Thus document WO 96/02841 describes a reagent for measuring haemoglobin without cyanide, which contains a detergent as well as a salt of hydroxylamine. This reagent can be used for numeration of the total leucocytes, but no leucocytic differentiation is possible.
Patent U.S. Pat. No. 5,242,832 describes a similar reagent which also permits partial leucocytic identification. However, this reagent does not permit identification of the basophil cells but permits solely evaluation of the lymphocytes, monocytes and granulocytes.
Document WO 98/32016 also describes a reagent of this type. However, the minority granulocytic subpopulations, namely the eosinophils and the basophils, are not identified by the reagent that is described in this publication.